Spinal muscular atrophy (SMA) is characterized by degeneration of the anterior horn cells of the spinal cord, leading to progressive symmetrical limb and trunk paralysis and muscular atrophy. SMA is the second most common fatal autosomal recessive disorder, second only to cystic fibrosis, and the most common genetic cause of childhood mortality affecting 1 in 6,000 newborns (Roberts et al., 1970, Arch. Dis. Child. 45:33-38; Pearn, 1973, J. Med. Genet. 10:260-265; Pearn, 1978, J. Med. Genet. 15:409-413; Czeizel and Hamular, 1989, J. Med. Genet. 21:761-763). Childhood spinal muscular atrophies are divided into severe (type I, Werdnig-Hoffman disease) and mild forms (type II and III) according to the age of onset and the severity of the disease (Munsat, 1991, Neuromusc. Disord. 1:81; Crawford and Pardo, 1996, Neurobiol. Dis. 3:97-110). The Survival of Motor Neurons (SMN) gene (Lefebvre et al., 1995, Cell 89:155-165) has been shown to be the SMA disease gene, and it is deleted or mutated in over 98% of SMA patients (Bussaglia et al., 1995, Nat. Genet. 11:335-337; Chang et al., 1995, Am. J. Hum. Genet. 57:1503-1505; Cobben et al., 1995, Am. J. Hum. Genet. 57:805-808; Hahnen et al., 1995, Hum. Mol. Genet. 4:1927-1933; Hahnen et al., 1996, Am. J. Hum. Genet. 59:1057-1065; Lefebvre et al., 1995, Cell 89:155-165; Rodrigues et al., 1995, Hum. Mol. Genet. 4:631-634; Velasco et al., 1996, Hum. Mol. Genet. 5:257-263; Lefebvre et al., 1997, Nat. Genet. 16:265-269).
Two inverted gene copies of the SMN gene are located in a 500 kb inverted repeat at chromosome 5q13. In over 98% of all SMA patients, the telomeric copy of SMN (SMNT) is deleted or mutated while the centromeric copy of the gene (SMNC) is unaffected (Lefebvre et al., 1995, Cell 89:155-165).
The SMN gene encodes a protein of about 296 amino acids having a molecular mass of approximately 40 kDa. The sequence of the protein does not exhibit any significant homology to any other protein of known function in the currently available protein databases.
Recently, in the course of studies of the functions of heterogeneous nuclear ribonucleoproteins (hnRNPs) (Dreyfuss et al., 1993, Ann. Rev. Biochem. 62:289-321), it was found that the SMN protein interacts with fibrillarin, an RNA-binding protein involved in rRNA processing, and with several other RNA-binding proteins (Liu and Dreyfuss, 1996, EMBO J. 15:3555-3565). Monoclonal antibodies to SMN localized the protein to a unique cellular location. SMN exhibits a general localization in the cytoplasm and is particularly concentrated in several prominent nuclear bodies called gems (for gemini of coiled bodies). Gems are novel nuclear structures which are related in number and size to coiled bodies and are usually found in close proximity to them (Liu and Dreyfuss, 1996, EMBO J. 15:3555-3565). Coiled bodies, which were first described by Ramxc3x3n y Cajal (1903, Trab. Lab. Invest. Biol. 2:129-221), are prominent nuclear bodies found in widely divergent organisms, including plant and animal cells (Bohmann et al., 1995, J. Cell Sci. 19:107-113; Gall et al., 1995, Dev. Genet. 16:25-35). Coiled bodies contain the spliceosomal U1, U2, U4/U6, and U5 snRNPs, U3 snoRNAs, and several proteins, including the specific marker p80-coilin, fibrillarin, and NOP140 (Bohmann et al., 1995, J. Cell Sci. 19:107-113, and references therein; Gall et al., 1995, Dev. Genet. 16:25-35). Expression of p80-coilin mutants and microscopic observations suggests a close association between coiled bodies and the nucleolus (Raska et al., 1990, J. Struct. Biol. 104:120-127; Andrade et al., 1991, J. Exp. Med. 173:1407-1419; Bohmann et al., 1995, J. Cell Biol. 131:817-831). However, the specific functions of coiled bodies are not clear. Current ideas propose that coiled bodies may be involved in processing, sorting, and assembly of snRNAs and snoRNAs in the nucleus. The close association of gems and coiled bodies raises the possibility that the SMN protein and gems are also involved in the processing and metabolism of small nuclear RNAs (Liu and Dreyfuss, 1996, EMBO J. 15:3555-3565).
The Sm class of small nuclear ribonucleoproteins (snRNPs) U1, U2, U4/6, and U5 are major constituents of the spliceosome, the catalytic center of the pre-mRNA splicing reaction (Moore et al., 1993, In: The RNA World, pp. 303-358, Gesteland and Atkins, eds., Cold Spring Harbor Laboratory Press, Plainview, N.Y.; Madhani and Guthrie, 1994, Annu. Rev. Genet. 28:1-26). Each spliceosomal snRNP consists of one (U1, U2, and U5) or two (U4/6) snRNAs, a common set of at least eight Sm proteins, termed B, Bxe2x80x2, D1, D2, D3, E, F, and G, and specific polypeptides that are associated with only one individual U snRNP (reviewed by Lxc3xchrmann et al., 1990, Biochim. Biophys. Acta Gene Struct. Express. 1087:265-292). With the exception of U6, all spliceosomal snRNAs share two structural features: the 5xe2x80x2-terminal trimethylguanosine (m3G) cap and a short, single-stranded, eight-to-ten nucleotide uridine-rich sequence flanked by two hairpin loops, referred to as the Sm site (Branlant et al., 1982, EMBO J. 1: 1259-1265; Reddy and Busch, 1988, In: Structure and Function of Major and Minor Small Nuclear Ribonucleoprotein Particles, pp. 1-37, Birnstiel, ed., Springer-Verlag, Berlin). The Sm site is the primary binding site for the Sm proteins. The remaining snRNA domains provide binding sites for the snRNA-specific snRNP proteins and for RNA-RNA interactions (Lxc3xchrmann et al., 1990, Biochim. Biophys. Acta Gene Struct. Express. 1087:265-292). U6 differs from the other spliceosomal U snRNAs in that it contains a xcex3-monomethyl cap instead of the (m3G) cap and does not bind directly to Sm proteins due to its lack of an Sm site (Reddy and Busch, 1988, supra; Singh and Reddy, 1989, Proc. Natl. Acad. Sci. USA 86:8280-8283). The snRNP-specific proteins have snRNP-specific functions in the splicing reaction. In contrast, the only known function for the Sm proteins is in the biogenesis of U snRNPs.
The biogenesis of snRNPs, which is illustrated in FIG. 26 herein, is a complex, multistep process (DeRobertis, 1983, Cell 32:1021-1025; Fisher et al., 1985, Cell 42:751-758; Mattaj, 1988, In: Structure and Function of Major and Minor Small Nuclear Ribonucleoprotein Particles, pp. 100-114, Birnstiel, ed., Springer-Verlag, Berlin; Feeney et al., 1989, J. Biol. Chem. 264:5776-5783; Neuman de Vegvar and Dahlberg, 1990, Mol. Cell. Biol. 10:3365-3375; Zieve and Sauterer, 1990, Crit. Rev. Biochem. Mol. Biol. 25:1-46). Spliceosomal snRNAs that contain the Sm site are first exported to the cytoplasm, where they associate with the Sm proteins (B, Bxe2x80x2, D1, D2, D3, E, F, and G) (Mattaj and DeRobertis, 1985, Cell 40:111-118). Next, in a reaction that requires the assembled Sm core domain (comprising the Sm proteins bound to the Sm site), the 7-methylguanosine (m7G) cap of the snRNAs is hypermethylated to yield 2,2,7-trimethylguanosine (m3G) (Mattaj, 1986, Cell 46:905-911). In addition, varying numbers of nucleotides are trimmed from the 3xe2x80x2 end of several of the snRNAs. Proper Sm core assembly, cap hypermethylation, and 3xe2x80x2-end processing are important for nuclear import of the assembled snRNP particles (Fischer and Lxc3xchrmann, 1990, Science 249:786-790; Hamm et al., 1990, Cell 62:569-577). Finally, just before or after the nuclear import, many internal nucleotides are modified and more than 30 snRNP-specific proteins associate with the individual snRNP precursors to complete their biogenesis (Mattaj, 1988, In: Structure and Function of Major and Minor Small Nuclear Ribonucleoprotein Particles, pp. 100-114, Birnstiel, ed., Springer-Verlag, Berlin; Lxc3xchrmann et al., 1990, Biochim. Biophys. Acta Gene Struct. Express. 1087:265-292; Neuman de Vegvar and Dahlberg, 1990, Mol. Cell. Biol. 10:3365-3375; Zieve and Sauterer, 1990, Crit. Rev. Biochem. Mol. Biol. 25:1-46). However, the detailed mechanism of how the Sm core proteins and the snRNP-specific proteins form functional assembled snRNPs is not clear.
There is, to date, no effective treatment for SMA and the mechanism underlying the disease process is poorly understood. Thus, there is an acute and long-felt need to understand the mechanism of the disease process and, more importantly, for the development of methods of treating this common and usually fatal disease. The present invention addresses these needs.
The invention relates to an isolated nucleic acid encoding a eukaryotic SIP1, and any mutants, derivatives, variants, and fragments thereof.
In one aspect, the isolated nucleic acid shares at least about 20% homology with at least one of huSIP1 (SEQ ID NO:1) and XeSIP1 (SEQ ID NO:3).
In another aspect, the isolated nucleic acid is selected from the group consisting of (SEQ ID NO:1), and (SEQ ID NO:3).
The invention further relates to an isolated nucleic acid encoding a eukaryotic SIP1, wherein the SIP1 shares at least about 20% homology with at least one of huSIP1 (SEQ ID NO:2), and XeSIP1 (SEQ ID NO:4), and any mutants, derivatives, variants, and fragments thereof.
Also included in the invention is an isolated polypeptide comprising a eukaryotic SIP1, and any mutants, derivatives, variants, and fragments thereof.
In one aspect, the SIP1 shares at least about 20% homology with at least one of SEQ ID NO:2 and SEQ ID NO: 4.
In another aspect, the amino acid sequence of the SIP1 is at least one of SEQ ID NO:2 and SEQ ID NO:4.
In another aspect, the nucleic acid further comprises a nucleic acid encoding a tag polypeptide covalently linked thereto.
In one aspect, the tag polypeptide is selected from the group consisting of a myc tag polypeptide, a myc-pyruvate kinase tag polypeptide, a His6 tag polypeptide, an influenza virus hemagglutinin tag polypeptide, a maltose binding protein tag polypeptide, and a glutathione-S-transferase tag polypeptide.
In another aspect, the nucleic acid of the invention further comprises a nucleic acid encoding a promoter/regulatory sequence operably linked thereto.
In yet another aspect, the nucleic acid further comprises a nucleic acid a tag polypeptide.
The invention also includes a cell comprising the nucleic acid of the invention.
In one embodiment, the cell is a DT40 cell.
Also included is a vector comprising the isolated nucleic acid of the invention.
In one aspect, the vector further comprises a nucleic acid encoding a promoter/regulatory sequence operably linked thereto.
Also included is a recombinant cell comprising the isolated nucleic acid of the invention.
In one aspect, the recombinant cell comprises the aforementioned vector.
In addition, the invention relates to an antisense isolated nucleic acid complementary to the nucleic acid of the invention.
Further, the invention relates to a cell comprising the antisense nucleic acid of the invention.
In addition, there is included an antibody that specifically binds to a eukaryotic SIP1 polypeptide, or a fragment thereof.
The antibody may be selected from the group consisting of a polyclonal antibody, a monoclonal antibody, and a synthetic antibody.
In a preferred embodiment, the antibody is a monoclonal antibody selected from the group consisting of 2S7 and 2E17.
The invention also relates to an isolated nucleic acid encoding a mammalian Gemin3, and any mutants, derivatives, variants, and fragments thereof.
In one aspect, the nucleic acid shares at least about 20% homology with human Gemin3 (SEQ ID NO:7).
In another aspect, the isolated nucleic acid is SEQ ID NO:7.
The invention further relates to an isolated nucleic acid encoding a mammalian Gemin3, wherein the Gemin3 shares at least about 20% homology with human Gemin3 (SEQ ID NO:8), and any mutants, derivatives, variants, and fragments thereof.
In addition, there is included an isolated polypeptide comprising a mammalian Gemin3, and any mutants, derivatives, variants, and fragments thereof.
In one aspect, the Gemin3 shares at least about 20% homology with SEQ ID NO:8.
In another aspect, the Gemin3 is SEQ ID NO:8.
In another aspect, the nucleic acid further comprises a nucleic acid encoding a tag polypeptide covalently linked thereto.
In one embodiment, the tag polypeptide is selected from the group consisting of a myc tag polypeptide, a myc-pyruvate kinase tag polypeptide, a His6 tag polypeptide, an influenza virus hemagglutinin tag polypeptide, a maltose binding protein tag polypeptide, and a glutathione-S-transferase tag polypeptide.
In another embodiment, the nucleic acid further comprises a nucleic acid encoding a promoter/regulatory sequence operably linked thereto.
Also included is a vector comprising the just-mentioned nucleic acid.
The vector may further comprise a nucleic acid encoding a promoter/regulatory sequence operably linked thereto.
In addition, the invneiton includes a recombinant cell comprising the just-mentioned nucleic acid.
The invention also includes a recombinant cell comprising the just-mentioned vector.
The invention further includes an antisense isolated nucleic acid complementary to the just-mentioned nucleic acid, and a cell comprising the same.
In addition, the invention relates to an nntibody that specifically binds to a mammalian Gemin3 polypeptide, or a fragment thereof
In one aspect, the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, and a synthetic antibody.
In one embodiment, the antibody is a monoclonal antibody selected from the group consisting of 11G9 and 12H12.
Also included is an antibody that specifically binds to a eukaryotic Survival of Motor Neurons (SMN) polypeptide, or a fragment thereof.
In one aspect, the SMN is human SMN and further wherein the antibody is monoclonal antibody 2B1.
In another embodiment, the SMN is chicken SMN.
In addition, the invention includes an isolated nucleic acid encoding a eukaryotic SIP1, and any mutants, derivatives, variants, and fragments thereof., wherein the nucleic acid comprises a mutation that affects binding of SIP1 with SMN.
Further, the invention includes an isolated nucleic acid encoding human SMN, wherein the nucleic acid comprises a mutation which mutation affects binding of SMN with at least one of another SMN protein, a Gemin3 protein, and an SIP1 protein.
In addition, the invention includes nn isolated nucleic acid encoding the human SMN protein, wherein the nucleic acid comprises a mutation which mutation affects pre-mRNA splicing.
The invention further includes a cell comprising the just-mentioned nucleic acid.
The invention also includes an isolated nucleic acid encoding a eukaryotic SIP1, and any mutants, derivatives, variants, and fragments thereof, wherein the nucleic acid comprises a mutation which mutation affects binding of SIP1 with SMN.
The invention also includes a fusion protein comprising a tag polypeptide and at least a portion of an SMN protein.
In one aspect, the tag polypeptide is selected from the group consisting of a myc tag polypeptide, a myc-pyruvate kinase tag polypeptide, a His 6 tag polypeptide, an influenza virus hemagglutinin tag polypeptide, a maltose binding tag polypeptide, and a glutathione-S-transferase tag polypeptide.
The invention further includes a fusion protein comprising a tag polypeptide and at least a portion of an SIP1 protein.
In addition, the invention includes a fusion protein comprising a tag polypeptide and at least a portion of a Gemin3 protein.
The invention also includes a vector comprising a nucleic acid encoding human SMN wherein the nucleic acid comprises a mutation and further wherein the mutation affects SMN binding to at least one of another SMN protein, a Gemin3 protein, and an SIP1 protein.
The invention also includes a composition comprising an isolated purified SMN protein and a protein that binds specifically with SMN.
In one aspect, the protein that binds specifically with SMN is selected from at least one of another SMN protein, an SIP1 protein, a Gemin3 protein, and an Sm protein.
In one embodiment, the composition further comprising a ribonucleic acid.
The invention further relates to a method of stimulating snRNP assembly. The method comprises contacting an extract comprising snRNP components with SMN, thereby stimulating snRNP assembly.
Also included is a mammalian cell comprising an exogenous SMN modulating sequence selected from the group consisting of a nucleic acid encoding SMN, an antisense nucleic acid complementary to a nucleic acid encoding SMN, and a ribozyme specific for ribonucleic acid encoding SMN, wherein the cell exhibits higher or lower levels of SMN protein compared with an otherwise identical cell which does not comprise the exogenous SMN modulating sequence.
In one embodiment, the exogenous SMN modulating sequence is an antisense nucleic acid and further wherein the cell exhibits a lower level of SMN protein compared with an otherwise identical cell which does not comprise the antisense nucleic acid.
In another embodiment, the cell further exhibits an altered growth characteristic compared with an otherwise identical cell which does not comprise the antisense nucleic acid.
In yet another embodiment, the exogenous SMN modulating sequence is a nucleic acid encoding SMN wherein the nucleic acid encoding SMN is covalently linked to a nucleic acid encoding a HA tag polypeptide, and further wherein expression of the exogenous SMN modulating sequence inhibits expression of endogenous SMN.
Also included is a method of identifying a compound which affects the level of SMN expression in a cell. The method comprises contacting the cell with a test compound and comparing the level of SMN expression in the cell with the level of SMN expression in a otherwise identical cell which is not contacted with the test compound, wherein a higher or lower level of SMN expression in the cell contacted with the compound compared with the level of SMN expression in the otherwise identical cell which is not contacted with the compound is an indication that the compound affects the level of SMN protein in the cell.
In one aspect, the compound increases the level of SMN expression in a cell.
In another aspect, the cell is obtained from a SMA type I patient.
In a further aspect, the cell is selected from the group consisting of a fibroblast and a lymphoblastoid cell.
In addition, the invenition includes a method of identifying a test compound which is a candidate SMA therapeutic. The method comprises contacting a cell with a test compound and comparing the level of SMN expression in the cell with the level of SMN expression in an otherwise identical cell which is not contacted with the test compound, wherein a lower level of SMN expression in the cell contacted with the test compound compared with the level of SMN expression in the otherwise identical cell which is not contacted with the test compound is an indication that the test compound is a candidate SMA therapeutic, thereby identifying a compound which is a candidate SMA therapeutic.
In one aspect, the cell is obtained from a SMA type I patient.
In another aspect, the cell is selected from the group consisting of a fibroblast and a lymphoblastoid cell.
The invention further includes a method of identifying a compound which affects the level of SMN expression in a cell comprising an SMN modulating sequence. The method comprises contacting the cell with a test compound and comparing the level of SMN expression in the cell with the level of SMN expression in an otherwise identical cell which is not contacted with the test compound, wherein a higher or lower level of SMN expression in the cell contacted with the compound compared with the level of SMN expression in the cell which is not contacted with the compound is an indication that the compound affects the level of SMN expression in the cell.
In one aspect, the SMN modulating sequence is selected from the group consisting of an isolated nucleic acid encoding SMN, an antisense nucleic acid complementary to a nucleic acid encoding SMN, and a ribozyme specific for ribonucleic acid encoding SMN.
In another aspect, the SMN modulating sequence is an antisense nucleic acid complementary to a nucleic acid encoding SMN.
Also included in the invention is a method of identifying a compound useful for the treatment of SMA. The method comprises contacting a cell comprising an antisense nucleic acid complementary to a nucleic acid encoding SMN with a test compound and comparing the level of SMN expression in the cell with the level of SMN expression in an otherwise dentical cell which is not contacted with the test compound, wherein a higher level of SMN expression in the cell contacted with the compound compared with the level of SMN expression in the cell which is not contacted with the compound is an indication that the compound is useful to treat SMA, thereby identifying a compound useful for the treatment of SMA.
In addition, the invention relates to a method of assessing whether a test compound affects binding of SMN with a protein that specifically binds with SMN. The method comprises (a) making a first preparation comprising a surface having at least a portion of SMN bound thereon, the test compound, and a labeled protein that specifically binds with SMN; (b) assessing the amount of the labeled protein bound with the surface in the first preparation; and (c) comparing the amount of the labeled protein bound with the surface in the first preparation and the amount of labeled protein bound with the surface in an otherwise identical preparation to which the test compound is not added, whereby a difference between the amount of labeled protein bound with the surface in the first preparation and in the otherwise identical preparation is an indication that the test compound affects the binding of SMN with a protein that specifically binds with SMN.
In one aspect, the protein that specifically binds with SMN is selected from the group consisting of another SMN protein, a SIP1 protein, a Gemin3 protein, a SmB protein, a SmBxe2x80x2 protein, a SmD1 protein, a SmD2 protein, and a SmD3 protein.
The invention further relates to a method of assessing whether a test compound is useful for treatment of SMA. The method comprises (a) making a first preparation comprising a surface having at least a portion of SMN bound thereon, the test compound, and a labeled protein that specifically binds with SMN; (b) assessing the amount of the labeled protein bound with the surface in the first preparation; and (c) comparing the amount of the labeled protein bound with the surface in the first preparation and the amount of labeled protein bound with the surface in an otherwise identical preparation to which the test compound is not added, whereby a lower amount of the labeled protein bound with the surface in the first preparation and in the otherwise identical preparation is an indication that the test compound is useful for treatment of SMA.
In one aspect, the protein that specifically binds with SMN is selected from the group consisting of another SMN protein, a SIP1 protein, a Gemin3 protein, a SmB protein, a SmBxe2x80x2 protein, a SmD1 protein, a SmD2 protein, and a SmD3 protein.
Also included is a method of enhancing splicing of mRNA. The method comprises incubating an in vitro pre-mRNA processing extract in the presence of SMN, or any mutant, derivative, variant, and fragment thereof, thereby enhancing splicing of the mRNA.
In addition, the invention includes a method of identifying a compound that affects pre-mRNA splicing. The method comprises incubating an extract capable of pre-mRNA splicing in the presence or absence of a test compound and comparing the level of pre-mRNA splicing in the extract in the presence of the test compound with the level of splicing of pre-mRNA in the absence of the test compound, wherein a higher or a lower level of pre-mRNA splicing in the extract in the presence of the test compound, compared with the level of pre-mRNA splicing in the extract in the absence of the test compound, is an indication that the test compound affects pre-mRNA splicing.
In addition, the invention relates to a method of identifying a test compound that is useful to treat SMA. The method comprises incubating an extract capable of pre-mRNA splicing in the presence or absence of a test compound and comparing the level of pre-mRNA splicing in the extract in the presence of the test compound with the level of splicing of pre-mRNA in the absence of the test compound, wherein a higher level of pre-mRNA splicing in the extract in the presence of the test compound, compared with the level of pre-mRNA splicing in the extract in the absence of the test compound, is an indication that the test compound is useful to treat SMA.
The invention further relates to a method of identifying a compound that affects snRNP assembly. The method comprises incubating an extract capable of snRNP assembly in the presence or absence of a test compound and comparing the level of snRNP assembly in the extract in the presence of the test compound with the level of snRNP assembly in the absence of the test compound, wherein a higher or a lower level of snRNP assembly in the extract in the presence of the test compound, compared with the level of snRNP assembly in the extract in the absence of the test compound, is an indication that the test compound affects snRNP assembly.
In addition, there is provided a method of identifying a test compound that is useful to treat SMA. The method comprises incubating an extract capable of snRNP assembly in the presence or absence of a test compound and comparing the level of snRNP assembly in the extract in the presence of the test compound with the level of snRNP assembly in the absence of the test compound, wherein a higher level of snRNP assembly in the extract in the presence of the test compound, compared with the level of snRNP assembly in the extract in the absence of the test compound, is an indication that the test compound is useful to treat SMA.
The invention further includes a method of assessing the presence or degree of SMA in a mammal. The method comprises obtaining a biopsy comprising motor neurons from the mammal and assessing the number and morphology of gems in the motor neurons, wherein a lower number of gems in the motor neurons, compared with the number of gems in motor neurons obtained from an otherwise identical mammal which does not have SMA, is an indication that the mammal has SMA, and further wherein the absence of or the presence of a minimal number of gems in the mammal having SMA is directly related to the severity of the SMA in the mammal.
The is further provided in the invention a method of assessing the presence or degree of SMA in a mammal. The method comprises comparing the level of binding of SMN obtained from the mammal to a protein that specifically binds with SMN with the level of binding of SMN wild type to an identical protein that specifically binds with SMN, wherein a lower level of binding of the SMN from the mammal to the protein that specifically binds with SMN compared with the level of binding of SMN wild type with the identical protein that specifically binds with SMN is an indication of the presence or degree of SMA in a mammal.
In one aspect, the protein that specifically binds with SMN is selected from the group consisting of an SMN protein, an SIP1 protein, and a Gemin3 protein.
The invention additionally includes a knock-out targeting vector, the vector comprising a first nucleic acid portion encoding a sequence 5xe2x80x2 of the open reading frame encoding SMN and a second nucleic acid portion encoding a nucleic acid sequence 3xe2x80x2 of the open reading frame encoding SMN.
In one aspect, the SMN is chicken SMN (SEQ ID NO:9).
In another aspect, the vector further comprises a nucleic acid encoding a selectable marker covalently linked thereto.
In one aspect, the first and second nucleic acid portions flank the nucleic acid encoding the selectable marker.
Also included is a recombinant cell comprising the aforementioned knock-out targeting vector.
The cell amy further comprise a vector comprising an isolated nucleic acid encoding SMN.
In one embodiment, the cell is a chicken pre-B lymphoid DT40 cell.
In addition, the invnetion includes a method of identifying a compound that affects SMN expression in a cell. The method comprises contacting the just-mentioned cell with a test compound and comparing the level of SMN expression in the cell with the level of SMN expression in an otherwise identical cell which is not contacted with the test compound, wherein a higher or lower level of SMN expression in the cell contacted with the test compound compared with the level of SMN expression in the otherwise identical cell which is not contacted with the compound is an indication that the compound affects SMN expression in a cell, thereby identifying a compound that affects SMN expression in a cell.
There is also provided a method of identifying a compound that is useful to treat SMA. The method comprises contacting the aforementioned cell with a test compound and comparing the level of SMN expression in the cell with the level of SMN expression in an otherwise identical cell which is not contacted with the test compound, wherein a higher level of SMN expression in the cell contacted with the test compound compared with the level of SMN expression in the otherwise identical cell which is not contacted with the compound is an indication that the compound increases SMN expression in a cell, thereby identifying a compound that is useful to treat SMA.
In addition, the invention includes a method of identifying a compound useful for the treatment of SMA. The method comprises contacting the aforementioned cell with a test compound and comparing the level of growth of the cell with the level of growth of an otherwise identical cell which is not contacted with the test compound, wherein a higher level of growth of the cell contacted with the compound compared with the level of growth of the cell which is not contacted with the compound is an indication that the compound is useful to treat SMA.
Also included in the invention is an isolated nucleic acid encoding a chicken SMN.
In one aspect, the nucleic acid shares at least about 20% homology with SEQ ID NO:9.
Further included is an isolated nucleic acid encoding chicken SMN, wherein the chicken SMN shares at least about 20% homology with SEQ ID NO:10.
In addition, the invention includes an isolated polypeptide comprising chicken SMN.
In one aspect, the SMN shares at least about 20% homology with SEQ ID NO:10.
In another aspect, the SMN is SEQ ID NO:10.